Pneumocystis carinii (Pc) is an opportunistic fungal pathogen causing pneumonia in immunocompromised individuals. Although considerable progress has been made in understanding the host immune response to the organism, fundamental questions regarding its biology still remain. This lack of knowledge is aggravated by, and contributes to, the inability to sustain continuous in vitro propagation of the organism. The development of a transformation system, allowing molecular manipulation of Pc, would represent a significant step in providing tools to study this pathogen. Identification of Pc genes by sequence homology, or Pc gene products by immunoreactivity has led to significant advances in the understanding of the organism. However, these techniques do not allow functional characterization of the gene products. The ability to introduce foreign DNA into Pc or to disrupt Pc genes may greatly advance our ability to manipulate the organism. An aggressive approach to understanding the biology of the organism would enhance understanding of the pathophysiology of infection, allow further development of the tools necessary to study and maintain the organism, and lead to novel methods of therapy of the infection. The overall goal of this research proposal is to develop a genetic transformation system for Pc. To achieve this goal, the aims of the research are 1) to establish a basic transformation system; 2) to characterize the fate of transforming DNA.